Aging-dependent changes in the effect of daily melatonin supplementation on rat metabolic and behavioral responses.

Pineal melatonin secretion has been reported to commonly decrease with aging, whereas intra-abdominal adiposity, plasma insulin and plasma leptin levels tend to increase. We recently demonstrated that daily melatonin administration starting at middle age suppressed male rat intra-abdominal fat, plasma leptin and plasma insulin to youthful levels, suggesting that aging-related changes in pineal melatonin secretion and in energy regulation may be functionally related. Accordingly, we have now investigated the effects of daily melatonin treatment on energy regulation in young versus middle-aged male Sprague Dawley rats. Addition of melatonin to the drinking water (0.2 microg/mL) produced nocturnal and diurnal plasma melatonin concentrations in middle-aged rats (12 months) equivalent to those of young adult (5 months) rats. Administration of this melatonin dosage every day for 10 wk starting at 10 months of age suppressed (P < 0.01) relative intra-abdominal fat, non-fasted plasma insulin and plasma leptin by 27, 39, and 51%, respectively (vs. vehicle-treated controls). In contrast, administration of melatonin for 10 wk starting at 3 months of age did not significantly alter (P> 0.10) any of these parameters. The melatonin administration stimulated (102%, P < 0.001) behavioral responsiveness of the middle-aged rats in a test of response to novelty, restoring youthful levels, but did not significantly alter behavioral responsiveness of the young rats. These results suggest that suppression of intra-abdominal adiposity and plasma leptin and insulin levels and stimulation of behavioral responsiveness in response to daily exogenous melatonin begins at middle age, coincident with and likely dependent upon the aging-associated decline in endogenous pineal melatonin secretion. These results further suggest that appropriate melatonin supplementation may potentially provide therapy or prophylaxis not only for the insulin resistance, increased intra-abdominal fat and resulting pathologies that occur with aging, but also for some aging-associated behavioral changes.

Chronic daily ethanol and withdrawal: 1. Long-term changes in the hypothalamo-pituitary-adrenal axis.

BACKGROUND: Hypothalamo-pituitary-adrenal (HPA) function has been demonstrated to be compromised for weeks and even months after alcoholics cease ethanol consumption. Because nonalcoholic subjects with family history-associated increased risk for alcoholism also exhibit compromised HPA function, it is not clear whether defects in the HPA axis of abstinent alcoholics reflect a preexisting condition that may be responsible for increased risk for alcohol abuse versus a persisting adaptational change in response to prolonged alcohol abuse. Consequently, we investigated whether chronic daily ethanol consumption and withdrawal by male Sprague Dawley rats would induce persistent HPA changes consistent with those demonstrated in abstinent alcoholics. METHODS AND RESULTS: In an initial experiment in which ethanol (5%, w/v) was incrementally introduced to liquid diet over a 1 week period followed by 4 weeks of chronic ethanol consumption, not only ethanol-treated rats but also pair-fed control rats exhibited decreased (p < 0.05 vs. ad-libitum-fed controls) anterior pituitary pro-opiomelanocortin (POMC) mRNA concentrations and associated decreases in plasma corticosterone and adrenocorticotropin (ACTH) levels for at least 3 weeks after gradual withdrawal of ethanol from the diet. Pair-feeding-induced decreases (p < 0.05) in thymus and spleen weights suggested that the pair-fed controls were likely stressed in this model, probably in response to the marked and irregular suppression of liquid diet consumption immediately after introduction of ethanol. Consequently, a second model was developed in which ethanol was introduced to the liquid diet much more gradually (i.e., over 3 weeks). In contrast with the rapid ethanol-introduction model, this more prolonged ethanol introduction followed by 4 weeks of chronic daily ethanol consumption increased plasma corticosterone levels (p < 0.05), increased adrenal gland weight (p < 0.05), and decreased thymus and spleen weights (both p < 0.01) without altering any of these parameters in the pair-fed controls. Three weeks after gradual withdrawal of ethanol from the diet, anterior pituitary POMC mRNA concentrations were suppressed (p < 0.05) and thymus and spleen weights were increased (p < 0.05) versus both pair-fed and ad-libitum-fed controls, accompanied by trends for decreased basal plasma corticosterone and adrenal weights. CONCLUSIONS: Chronic daily ethanol treatment induced changes in the HPA axis that persisted for at least 3 weeks after complete cessation of ethanol consumption. These persistent alterations in the HPA axis are similar to the aberrant HPA regulation of abstinent alcoholics, sons of alcoholics, Lewis rats, and individuals who suffer from posttraumatic stress disorder and some types of depression, that is, categories of individuals who all exhibit increased risk for high ethanol consumption. Thus, these chronic daily ethanol-induced persistent changes in the HPA axis may have significant roles in alcohol abstinence syndrome and may increase vulnerability to relapse.

Enzyme-linked immunospot assay for the diagnosis of active Treponema pallidum infection during the various stages of syphilis.

BACKGROUND: Specific serologic assays for syphilis cannot differentiate current infections from past infections and are inefficient to monitor efficacy of antibiotic therapy. GOAL: To develop a new immunologic assay for the identification of active Treponema pallidum infection during the various stages of syphilis. STUDY DESIGN: Peripheral blood mononuclear cells obtained from patients with syphilis in an STD clinic were tested for T. pallidum-specific circulating antibody-secreting cells (ASC) by an enzyme-linked immunospot assay (ELISPOT). RESULTS: Specific ASC were demonstrated in all six patients with primary syphilis and in 14 of 16 patients diagnosed with secondary syphilis. ASCs were undetectable in five patients 8 to 16 days after appropriate therapy, but persisted in one case that was considered treatment failure. Among the 13 patients diagnosed with latent syphilis, six (46%) demonstrated ASC, reflecting antigenic stimulation. CONCLUSION: The ELISPOT assay is effective for the diagnosis of primary and secondary syphilis. The presence of circulating ASC suggests persistent active infection in some patients during the latent disease stage.

Quality assessment activities in Latin America and the Caribbean.

The Pan American Health Organization (PAHO) supports performance evaluation programs in clinical chemistry, Caribbean. Much more work remains to be done before many labs in this part of the world can be certified as providing accurate results, but PAHO has taken an important first step toward establishing benchmarks and rallying for labs to improve test quality.

Cross-reactivity to Borrelia burgdorferi proteins in serum samples from residents of a tropical country nonendemic for Lyme disease.

Reports of Lyme disease from areas where the disease is not endemic have increased. Eighty-six human serum samples from Papua New Guinea (nonendemic for Lyme disease) were examined for the presence of IgG antibodies that recognize Borrelia burgdorferi antigens, using the currently recommended two-tiered system of analysis (sensitive ELISA with Western blot). The percentage of positive tests dropped from 50% to 10% when individual negative controls were included in the two-tiered analysis. Positive serum samples failed to inhibit the growth of B. burgdorferi in culture and did not yield positive reactions in the fluorescent treponemal antibody-absorption test. These characteristics, together with atypical Western blot antigen recognition patterns and the absence of known vectors, provide evidence that seropositive results for these persons are not the result of exposure to B. burgdorferi. Individual negative controls may minimize false-positive results for serologic tests for Lyme disease, and these tests must be interpreted in the context of clinical and epidemiologic data.

Crack cocaine smoking and oral sores in three inner-city neighborhoods.

Crack cocaine causes blisters, sores, and cuts on the lips and in the mouths of persons who smoke it, and such sores may facilitate the oral transmission of HIV. We recruited young adults aged 18-29 years, who either were current regular crack smokers, or who had never smoked crack, from inner city neighborhoods in New York, Miami, and San Francisco. Participants were interviewed for HIV risk behaviors and history of recent oral sores and were tested for HIV, syphilis, and herpes simplex virus (HSV) antibodies. Among the 2,323 participants recruited, 1,404 (60%) were crack smokers. Crack smokers (10.0%) were more likely than nonsmokers (4.5%) to report having had oral sores in the past 30 days [prevalence odds ratio (POR) 2.4, 95% confidence interval (CI) 1.7-3.4]. Sores were also more prevalent among those who had ever injected drugs (14.3%) than among those who had not (6.7%; POR 2.3, 95% CI 1.7-3.4), and among those with HIV infection (14.3%) than among those without it (8.0%; POR 1.9, 95% CI 1.3-2.8). Among the 429 participants who reported receptive oral sex, those who reported oral sores were more likely than those who did not to have HIV infection, after other HIV risk factors were controlled for (adjusted POR 1.9, 95% CI 1.0-3.6). Our results confirm that crack smokers have a high prevalence of oral sores and provides evidence that these sores, although infrequently, may facilitate oral transmission of HIV.

Laboratory diagnosis of sexually transmitted diseases in facilities within the United States. Results of a national survey.

BACKGROUND AND OBJECTIVES: The diagnosis of many sexually transmitted diseases (STD) requires laboratory testing. The authors assessed the effects of the introduction of new tests and regulations on STD testing. STUDY DESIGN: A questionnaire survey was mailed to a random sample of facilities listed in the STD Referral Database inquiring about tests offered, changes in testing, and reasons for changes. RESULTS: Responses from 405 facilities were analyzed. Most responding facilities collected specimens for nontreponemal tests for syphilis (352 of 405 [86.9%]). Since each facility's information was last updated, the number reporting testing for Chlamydia trachomatis rose from 160 of 405 (39.5%) to 288 of 405 (71.1%), but testing for gonorrhea and chancroid decreased (365 of 405 [90.1%] to 328 of 405 [81%], and 182 of 405 [44.9%] to 32 of 405 [7.9%], respectively). Of 364 responses to a question on changes in tests performed in the last 2 years, 249 (68.4%) reported no change, 81 (22.3%) reported an increase, and 37 (10.2%) reported a decrease. The most frequently added tests were nonculture tests for C. trachomatis (34 of 81 [42%]) and the most frequent reason for adding tests was targeted funding (25 of 81 [30.9%]). The most frequently discontinued tests were cultures and gram stains for gonorrhea (15 of 37 [40.5%]) and other in-house tests (9 of 37 [24.3%]). Most facilities that discontinued testing cited the Clinical Laboratory Improvement Act as the reason (34 of 37 [91.9%]; 95% confidence interval = 78.1%, 98.3%). CONCLUSIONS: The number of facilities testing for C. trachomatis has increased with funding and with the availability of nonculture tests, but the number of those testing for chancroid and gonorrhea has decreased. Implementation of the Clinical Laboratory Improvement Act may be associated with a decrease in the number of facilities performing tests for STD.

Instrument-dependent fluorochrome sensitivity in flow cytometric analyses.

Flow cytometry has become the preferred technique by which critical clinical evaluations are made such as CD4 counts and aneuploid analyses. Mounting concern has arisen over the numerous techniques, reagents, and different flow cytometric employed to determine these data. Several studies have documented significant differences in results when different flow cytometers are utilized to analyze the same sample. Fluorochrome-dependent instrument sensitivity also has been reported by numerous investigators. As more and more procedures are performed by cytometric analysis, light scatter and fluorescence limitations, which appear to be instrument dependent, demonstrate that not all flow cytometers have the same capabilities. Attempts were made to calculate molecules of equivalent soluble fluorochrome (MESF) values on nine different flow cytometers using fluorescein isothiocyanate (FITC) and R-phycoerythrin (R-PE) labeled microsphere reference standards produced by Flow Cytometry Standards Corporation (FCSC). Dramatic differences were observed in the ability of some cytometers to resolve these microspheres. The diminished resolution appeared to be instrument model and fluorochrome dependent. We propose that diminished fluorescence resolution in certain flow cytometers could be responsible for significant variability in clinical values reported from laboratories utilizing different flow cytometers.

Pathology of the umbilical cord in congenital syphilis: analysis of 25 specimens using histochemistry and immunofluorescent antibody to Treponema pallidum.

Identification of Treponema pallidum in the placenta is important for diagnosis of congenital syphilis; however, spirochetes are difficult to observe in chorionic villi. To determine the sensitivity of umbilical cord examination for T pallidum, and the association of spirochetes with cord pathology, placentas were prospectively obtained from 25 women with untreated syphilis. The most common finding using hematoxylin-eosin staining was a normal-appearing umbilical cord (48%); necrotizing funisitis was the most frequent pathological lesion (36%). Spirochetes were detected using silver and immunofluorescent staining in 89% of cords, including 92% of histologically normal and 84% of abnormal cords. Three specimens showed subamnionic aggregates of spirochetes, consistent with amniotic fluid infection. Necrotizing funisitis was strongly associated with umbilical artery infection by spirochetes (P = .008). There was a 100% correlation between results of silver and immunofluorescent staining. The umbilical cord is a sensitive site for morphological confirmation of T pallidum; it is significant for the pathologist that spirochetes may often be detected in the absence of overt tissue inflammation or necrosis.

Laboratory diagnosis and interpretation of tests for syphilis.

The lack of a method for demonstrating the presence of Treponema pallidum by growth necessitates the use of alternative methods. Traditionally, these methods are divided into direct detection methods (animal inoculation, dark-field microscopy, etc.) and serologic tests for the presence of patient antibody against T. pallidum. Serologic methods are further divided into two classes. One class, the nontreponemal tests, detects antibodies to lipoidal antigens present in either the host or T. pallidum; examples are the Venereal Disease Research Laboratory and rapid plasma reagin and tests. Reactivity in these tests generally indicates host tissue damage that may not be specific for syphilis. Because these tests are easy and inexpensive to perform, they are commonly used for screening, and with proper clinical signs they are suggestive of syphilis. The other class of test, the treponemal tests, uses specific treponemal antigens. Confirmation of infection requires a reactive treponemal test. Examples of the treponemal tests are the microhemagglutination assay for antibodies to T. pallidum and the fluorescent treponemal antibody absorption test. These tests are more expensive and complicated to perform than the nontreponemal tests. On the horizon are a number of direct antigen, enzyme-linked immunosorbent assay, and PCR techniques. Several of these techniques have shown promise in clinical trials for the diagnosis of congenital syphilis and neurosyphilis that are presently difficult to diagnose.

Syphilitic endometritis causing first trimester abortion: a potential infectious cause of fetal morbidity in early gestation.

This report describes a 30-year-old woman with a history of prostitution and "crack" cocaine use during pregnancy. After an incomplete abortion at 11 weeks' gestation, a suction dilatation and curettage of uterine contents was done. Histopathologic examination revealed lymphoplasmacytic deciduitis and decidual necrosis, as well as rare first trimester chorionic villi. Silver staining using the Steiner technique showed numerous spirochetes, morphologically consistent with Treponema pallidum, in the decidualized endometrial tissues. This case of syphilitic endometritis, which appears to be the first reported, suggests that treponemal infection can cause fetal morbidity during early gestation.

Prevalence, incidence, and correlates of syphilis seroreactivity in multiethnic San Francisco neighborhoods.

To examine the extent of infection with syphilis in an inner-city community, we determined the prevalence, incidence, and correlates of syphilis seroreactivity in a representative sample of unmarried whites, African Americans, and Hispanics living in San Francisco during 1988 to 1989 and again 1 year later in 1989 to 1990. One thousand seven hundred seventy single men and women aged 20 to 44 were surveyed in a random household sample drawn from three neighborhoods of varying geographic and cultural characteristics. Syphilitic infection was determined by testing specimens with the microhemagglutination assay for antibodies to Treponema pallidum (MHA-TP). Of blood samples available from 1262 participants from the initial survey, 32 (2.5%) were MHA-TP reactive. After adjustment for age, a reactive syphilis serology was significantly predicted (P < 0.05) by African American race, homosexual activity (men), and less education. In homosexually active men, lifetime number of male sex partners and the presence of antibody to the human immunodeficiency virus (HIV) significantly predicted syphilis seroreactivity (P < 0.01). One year later, of 841 specimens available for testing, an additional 13 (1.5%) had become MHA-TP reactive. Eleven (85%) of the new cases were in heterosexual men and women. Although San Francisco citywide incidence data indicate that syphilis may be decreasing for the city as a whole, incidence data on a community level suggests that syphilitic infection is increasing in high-risk heterosexual communities. Thus, syphilis prevention programs should rely on serologic testing at the community level to plan effective intervention strategies.

Flow cytometric analysis of peripheral blood lymphocyte immunophenotypes in persons infected with Treponema pallidum.

To characterize the human immune response to syphilis, we determined the effect of infection with Treponema pallidum on the percentage of the various lymphocyte subpopulations in the peripheral blood of infected and uninfected persons. Monoclonal antibodies labeled with either fluorescein isothiocyanate or phycoerythrin were used to perform dual color analysis on a FACScan with the following markers: CD3 for total T cells, CD4 for T helper cells, CD8 for T suppressor cells, CD19 for B cells, and CD16 plus CD56 for natural killer cells. Lymphocyte immunophenotype results were analyzed by the stage of untreated syphilis and by gender. Although they were within the ranges of the normal distribution of immunophenotypes, the percentages of CD4+ cells were significantly lower (P < 0.001) and those of CD8+ cells were higher (P = 0.03) in patients with syphilis than in the uninfected population. For infected versus uninfected subjects, both women and men, the differences in the mean percentages of CD3+ and CD4+ cells were significant (P < or = 0.05). Significant differences were noted between the sexes in secondary syphilis only in the mean percentages of cells positive for CD3, CD4, CD8, and CD16 plus CD56. Gender had no effect on lymphocyte subpopulations in subjects with primary or latent syphilis. In the control population, significant differences due to gender were observed in the percentages of cells positive for CD3, CD4, and CD16 plus CD56.

Comparison of the fluorescent treponemal antibody absorption (FTA-ABS) test with the FTA-ABS double staining test for detection of antitreponemal immunoglobulin M in the 19S fraction of human serum.

A widely used immunoglobulin M (IgM) detection assay for the diagnosis of neonatal congenital syphilis is the fluorescent treponemal antibody absorption test used with fractionated serum (FTA-ABS 19S IgM test). Reading the results of the FTA-ABS test is more cumbersome than reading those of the FTA-ABS double staining (FTA-ABS-DS) test, a confirmatory test for specific IgG. To verify that the FTA-ABS-DS test used with an anti-human IgM conjugate could detect specific IgM in fractionated serum samples (FTA-ABS-DS 19S IgM test), 164 fractionated (QUIK-SEP IgM Isolation System; ISOLAB, Inc., Akron, Ohio) serum specimens from infected neonates or adults or from IgG-seronegative subjects were tested by both techniques. The sensitivity limits of the two tests were assessed with reactive serum samples diluted to an endpoint titer. Samples nonreactive by the FTA-ABS 19S IgM test (n = 74) were either nonreactive (n = 65), minimally reactive (n = 5), or reactive (n = 4) by the FTA-ABS-DS 19S IgM test. Samples minimally reactive by the FTA-ABS 19S IgM test (n = 32) were minimally reactive (n = 1) or reactive (n = 31) by the double staining test. All samples reactive by the FTA-ABS 19S IgM test (n = 58) were also reactive by the FTA-ABS-DS 19S IgM test. There was a directly proportional linear relationship (r = 0.9794) between titers obtained by both tests. FTA-ABS-DS 19S IgM titers were constantly equal to or higher than FTA-ABS 19S IgM titers. Fluorescence intensity reading repeatability was 91.4% for the FTA-ABS-DS 19S IgM test and 81.7% for the FTA-ABS 19S IgM test (P = 0.015). Because the more easily read FTA-ABS-DS 19S IgM test is at least as sensitive as, if not more sensitive than, the FTA-ABS 19S IgM test, it is a good alternative to the latter test for the detection of specific IgM in human fractionated sera for those using fluorescence microscopes with incident light.

Seroepidemiological characterization of a syphilis epidemic in the Republic of the Marshall Islands, formerly a yaws endemic area.

The annual numbers of reported cases of syphilis in the Republic of the Marshall Islands (RMI) increased from none in 1983 to more than 600 in 1989, suggesting a large outbreak of syphilis. Much of the increase resulted from expanded serological screening. The apparent outbreak of syphilis, therefore, may have been partly the result of increased surveillance or, since the RMI was formerly a yaws endemic area, possibly due to a resurgence of yaws. To address this problem and better characterize the epidemic, we analysed results from a 1989/90 Ministry of Health Services mass serological screening on Majuro Atoll, the main population centre. Serum specimens from 9160 people (86% of residents aged 15-44 years) on Majuro were screened with the rapid plasma reagin (RPR) card test; we repeated the RPR and performed a confirmatory microhaemagglutination assay for Treponema pallidum-specific antibodies (MHA-TP) on a sample of serum specimens. To estimate the seroprevalence of syphilis, we also tested a sample of RPR nonreactive specimens by MHA-TP. Among people less than 45 years of age, total (11.5%) and high-titre (5.2%) seropositivity rates were highest in the 20-24 year age group, as was MHA-TP seroprevalence (15.9%). These results suggested that a large outbreak of syphilis was responsible for the observed seroreactivity. Cumulative incidence modelling and comparisons with the results of a previous serosurvey conducted in 1985 suggested that the duration of the syphilis epidemic was approximately 10 years and that incidence had not increased appreciably since 1985.

Specific immunofluorescent staining of pathogenic treponemes with a monoclonal antibody.

Two hybrid cell lines which produced mouse monoclonal antibody to the DAL-1 street strain of Treponema pallidum subsp. pallidum were established. These monoclonal antibodies strongly reacted with T. pallidum subsp. pallidum (Nichols strain, DAL-1, and two other street strains, strains MN-1 and MN-3) and T. pallidum subsp. pertenue by indirect microimmunofluorescent antibody and enzyme-linked immunosorbent assay techniques, but they did not react with normal rabbit testicular tissue. These monoclonal antibodies did not react with nonpathogenic treponemes, such as T. phagedenis Reiter, T. denticola MRB, T. refringens Noguchi, or other spirochetes, such as Borrelia burgdorferi and Leptospira interrogans serovar pomona in microimmunofluorescent antibody smear slides or in Western blots (immunoblots). While unlabeled antibodies are useful for investigating the antigenic structures of T. pallidum, we labeled these monoclonal antibodies with fluorescein isothiocyanate and used them for diagnosing syphilis by direct staining of lesion exudate or T. pallidum subsp. pallidum in formalin-fixed tissues from patients suspected of having syphilis. Both monoclonal antibodies were directed against antigens of T. pallidum subsp. pallidum with a molecular weight of 37,000 as determined by the Western blotting technique.

Anatomical evidence of synaptic plasticity in the cochlear nuclei of white-deaf cats.

Synapses are dynamic structures reflecting environmental events. It was observed that densities associated with apposing synaptic membranes were altered in response to changing conditions of auditory stimulation (Gulley et al. 1978. J. Comp. Neurol. 180: 707-742). Synapses also exhibit plasticity during early development of the auditory system (Larsen and Pappas, 1985. Proceedings: 43rd Annual Meeting of the Electron Microscopy Society of America, pp. 493-494). Larsen and Pappas reported that, in young kittens, the end bulb of Held (EBH) terminal was distinguished by excessive density at apposing synaptic membranes which assume gradually the pattern observed in adult cats. The maturation of synapses parallels emerging function and may depend upon auditory stimulation (Larsen and Kirchhoff, 1987. Neurosci. Abstr. 13: 1260). Larsen and Kirchhoff found that EBH synapses on large spherical cells of white-deaf cats resembled the immature synapses found in young kittens (Fig. 1). They also found terminals that had an increased number and length of the membrane densities associated with synapses. Because the number of synaptic vesicles in these presynaptic terminals was equivalent to the number of vesicles found in normal-hearing cats, Larsen and Kirchhoff (1987) suggest that this is evidence of synaptic plasticity. We have completed a quantitative study of the synapses of EBH terminals found in adult cats that had been deaf for at least 3 years.

A population-based serosurveillance of syphilis in Costa Rica.

As part of a case-control study to investigate the high incidence of cervical cancer in Costa Rican women, the seroprevalence of the treponematoses, in particular, syphilis was determined. In each age group, women with a history of two or more sex partners were two to four times more likely to be seroreactive in tests for syphilis than women with zero or one sex partner. The highest percentage of reactive results in the microhemagglutination assay for antibodies to Treponema pallidum (MHA-TP) was seen in samples from women aged 50-59 who had had two or more lifetime partners (23.8%). Three observations from our study support reactivity due to syphilis rather than yaws or pinta: (1) a similar percent of reactive rapid plasma reagin (RPR) card test results among MHA-TP reactors in the two age groups of women who were surveyed (42 vs. 49%) was observed; (2) women who were seroreactive in the MHA-TP had multiple risk factors for STD [low socioeconomic status (9.4%), urban residence (22.8%), first intercourse under 16 years of age (14.1%), and multiple sex partners (26.3%)], and (3) only sexually experienced women had reactive results in the MHA-TP test.